Clathrin-mediated endocytosis (CME) is required for the normal growth and development of all eukaryotes. It is an important route by which cells take up many nutrients and regulatory molecules and down-regulate membrane proteins in response to their ligands and environmental signals. Further importance of this pathway is exemplified by a number of human diseases that are associated with CME, including cancer, cardiovascular and neurological disorders, and entry of infectious viruses into cells. While many factors involved in CME have been identified and a large number of protein-protein or protein- lipid interactions for them have been described, assigning specific roles to these proteins remains a challenge to the field. There is still much to learn about how cargo are collected, the importance of the elaborate network of interactions and scaffolding functions of the endocytic machinery, how force is generated to invaginate and pinch off a vesicle, and how the tinning and regulation of assembly and disassembly of these components coordinate this dynamic process. Studies in this lab have taken advantage of the powerful molecular genetic methods of S. cerevisiae to analyze clathrin function. In yeast, as in all eukaryotes, both clathrin and the actin cytoskeleton are critical for RME. Furthermore, many of the endocytic factors identified in yeast have counterparts in animal cells that are involved in clathrin-mediated internalization, indicating that yeast can serve as an important model for studying clathrin and endocytosis. In the previous grant period live cell imaging methods were established to examine the dynamics of clathrin during endocytic vesicle formation in yeast. Clathrin was found to be one of the earliest factors recruited to endocytic sites, and this recruitment depends upon the yeast epsin and AP180-like adaptors. Also, a novel endocytic-specific function for clathrin light chain (LC) that involves binding to Sla2p (related to mammalian Hip1/R) to promote the progression of early endocytic patches to the invagination stage of vesicle formation was discovered. Finally, studies examined the role of a novel endocytic factor, Scd5, which binds type 1 protein phosphatase, and suggest that Scd5/PP1 is part of a phospho-regulatory cycle, wherein it may reactivate several early endocytic factors for new rounds of vesicle internalization. This proposal extends these studies to: (1) Examine in detail the requirements for and dynamics of formation of early endocytic sites using live cell imaging methods;(2) Examine how clathrin LC regulates the progression of early endocytic vesicle formation;and (3) Examine how ScdSp, as a targeting subunit for PP1, regulates the formation of early endocytic vesicles. These studies shall provide novel insight into how endocytic sites are established and how phosphorylation regulates this dynamic process.